Identification of the C9-hydrogenase for 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (9,17-DOHNA) and the 7α-dehydratase essential for initiating β-oxidation of the B-, C-, and D-rings in steroid degradation by <i>Comamonas testosteroni</i> TA441

Comamonas testosteroni TA441 is a model aerobic steroid-degrading bacterium whose sterane degradation pathway has been elucidated in the greatest detail to date. Similar pathways have been identified in many genera of bacteria, including both proteobacteria and actinobacteria, such as Mycobacterium tuberculosis . However, the genes encoding the C9-hydrogenase for 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (9,17-DOHNA, also known as HIP) and the 7α-dehydratase essential for initiating β-oxidation of the B-, C-, and D-rings had not been identified. In this study, we identified these missing genes, located adjacent to the chsE1E2H1H2ltp2 cluster involved in C17 side-chain degradation, and designated them scdB and scdH , respectively. This finding completes the elucidation of all degradation steps of 9,17-DOHNA prior to D-ring cleavage. AlphaFold models showed that ScdB and at least five hydrogenases/dehydrogenases involved in steroid degradation in TA441 share a similar dimer structures with Rossmann fold motif. In contrast, ScdH was predicted to form a homohexameric structure similar to ScdY and ScdN, involved in B-, C-, and D-ring degradation in TA441. Furthermore, AlphaFold modeling revealed that SteC, the dehydratase responsible for removing the C12β-hydroxyl group from 9,17-DOHNA derivatives, exhibits strong structural similarity to BaiE, the bile acid 7α-dehydratase of Clostridium scindens JCM 10418/VPI 12708, despite sharing only ~28% amino acid sequence identity.IMPORTANCEResearch on bacterial aerobic steroid degradation began more than 70 years ago, initially to produce intermediates for steroid drug synthesis. Recently, this field has gained renewed attention due to its implications for human health-for example, the role of cholesterol import and degradation in the persistence of Mycobacterium tuberculosis H37Rv within chronically infected lungs. Comamonas testosteroni TA441 serves as a key model organism for elucidating aerobic steroid degradation, with pathways for cleavage of the A-, B-, C-, and D-rings already well established. The functions and structures of the enzymes identified in TA441 display striking similarities to those in actinobacteria, such as M. tuberculosis . In this study, we identified two enzymes indispensable for initiating β-oxidation of the B-, C-, and D-rings, thereby filling the last remaining gaps for initiating this pathway. Our AlphaFold-based structural analysis of these enzymes not only provides new insights into the steroid metabolism of M. tuberculosis but also broadens understanding of the ecological and physiological significance of bacterial steroid degradation.